The ability of hybrid tumor cells to induce antitumor immunity has been evaluated in the line 1 alveolar cell carcinoma (L1) model of BALB/c mice. Hybrid tumor cells were produced by fusing freshly dissociated L1 cells isolated from in vivo tumors with the hypoxanthine:aminopterin:thymidine-sensitive cell line, GM 347, derived from C3H mice. Each hybrid was characterized by DNA content and expression of H-2 antigens using a fluorescence-activated cell sorter. Irradiated L1 cells in the presence or absence of Corynebacterium parvum were capable of immunizing BALB/c mice against a challenge of live L1 cells, provided the challenge dose was small (50% lethal dose was between 6 X 10(4) and 1.2 X 10(5) L1 cells). Testing of five hybrid clones and 1 uncloned hybrid line for their immunizing ability demonstrated a range in immunizing ability with none showing a statistically significant improvement in survival (p less than 0.0018) when compared to untreated controls. However, one hybrid clone, MoHb-L1-C2, was selected in which the survival of mice immunized with it compared to controls had a p value of 0.0255. A tumor (labeled L1/A) which grew in one of the mice immunized with this clone was removed and hybridized with GM 347 to yield a second set of hybrids. Both this variant of L1 cells and a hybrid clone made from it (MoHb-L1A-C18) were capable of immunizing mice against a challenge of live L1/A (p values of 0.0000 and 0.0028, respectively, when compared to controls). However, L1 cells were not able to immunize effectively against L1/A, and MoHb-L1A-C18 did not immunize against L1. This suggests that L1/A is a subpopulation of L1 cells with a different antigenic composition. The limited success of MoHb-L1A-C18 against L1/A is thought to be due to the narrower range of antigenic specificities in L1/A and the ability of MoHb-L1A-C18 to represent an important antigenic subpopulation of L1/A.